Species-specific DNA probes for Vibrio vulnificus and Vibrio cholerae, methods and kits

ABSTRACT

The present invention relates to species-specific DNA probes specific for  Vibrio vulnificus  and  Vibrio cholerae . The DNA probes of the present invention specifically detects  Vibrio vulnificus  or  Vibrio cholerae  in a mixed bacterial sample based on unique ribosomal RNA nucleotide sequences. When the DNA probes of the present invention are tagged with a labeled molecule such as a fluorescent label, it affords direct and immediate visualization of individual bacterial cells, and a rapid method of detection of bacterial infection in humans and shellfish without culturing.

This is a continuation of application Ser. No. 08/474,828 filed on Jun.7, 1995 abandoned, which is a division of Ser. No. 07/891,987, filed May28, 1992, U.S. Pat. No. 5,426,025.

FIELD OF THE INVENTION

The present invention relates to species-specific oligonucleotide probesfor binding specifically to ribosomal RNA of bacterium Vibrio Vulnificusor Vibrio cholerae. The present invention further relates to methods andkits for identifying the bacteria Vibrio vulnificus or Vibrio choleraewith such probes in a single day, without the need for culturing thebacteria.

BACKGROUND OF THE INVENTION

Vibrio vulnificus and Vibrio cholerae are small organisms calledbacteria that live in the marine environment. Vibrio cholerae can alsosurvive in fresh water. By drinking water, eating fruits and vegetables,fish or shellfish that are contaminated with this bacterium (onebacteria), a person can become very ill or may even die from the diseasecholera which causes severe diarrhea and dehydration. Vibrio vulnificuscan cause serious illness and even death within three days in people whoeat raw or improperly cooked fish or shellfish that are infected withthis microorganism.

There are many types of bacteria, both good and bad, in food and water.To find out whether water or food contains these harmful Vibriobacteria, or if a person is infected with them, laboratory tests must beperformed. The first step is to culture, or grow, the bacteria in aspecial liquid. Then a series of tests are done to identify the bacteriabased on whether or not they use certain sugars and other compounds inorder to grow. It may take as long as one week to do these tests and bythat time a person may die if not given the proper medicine. What isneeded is a rapid and easy way to detect and identify Vibrio choleraeand Vibrio vulnificus.

A faster way to check for the presence of these bacteria is to use amolecule called an antibody that recognizes then binds to a specificpart of the bacterium. See, for example, Tamplin, M. L. et al.: Appliedand Environmental Microbiology, 57:1235-1240 (April 1991), which reportsan enzyme immunoassay for the identification of Vibrio vulnificus inseawater, sediment and oysters. However, this method also requiresgrowing the bacteria first and still takes several days to do. Also,antibodies may be too specific and detect only those bacteria from onesource but not from another.

Another method involves the use of a “gene probe” to identify thebacteria. Every living organism has molecules of DNA (deoxyribonucleicacid) which contains instructions for each cell to make the things itneeds to sustain life. A gene is a piece of DNA that instructs the cellto make one particular type of protein molecule. A gene probe is alsomade of DNA. See, for example, Amann, R. et al.: Nature. 351:161-164(May 9, 1991); and Amann, R. et al.: J. Bacteriology. 173:762-770(February 1990).

Vibrio vulnificus and Vibrio cholerae have specific genes that makeproteins which are responsible for causing sickness. A gene probe thatspecifically seeks out the genes for these toxic proteins can be used tofind out whether these bacteria are in food, water or people. However,there is a problem with using a gene probe. Bacteria are one-celledorganisms. Each bacterium has only one gene for each kind of protein itmakes. It is difficult to detect the one and only toxic protein gene ineach cell.

One way to solve this problem is to use a method called PCR to amplify(make many copies of) the gene in a test tube and then use the geneprobe to find the gene copies. This method, however, is sophisticatedand time-consuming and requires a sufficient number of bacteria toperform the method.

With the current outbreak of Vibrio cholerae in South America, it isapparent that a rapid means of detection is warranted. Consequently,there is a need in the industry for simple, but quick and accurate testsfor the reliable and early detection of the bacteria Vibrio vulnificusor Vibrio cholerae in, for example, water, food, blood, feces, and thelike, contaminated with such bateria.

SUMMARY OF THE INVENTION

In brief, the present invention alleviates and overcomes certain of theabove-referenced problems and shortcomings of the present state of theart through the discovery of novel oligonucleotide probes which arespecies-specific for the 23S rRNA of Vibrio vulnificus and Vibriocholerae. Generally speaking, the DNA probes of the present inventionseek out and hybridize with specific, unique RNA regions of each 23Sribosome in every cell of either the bacteria Vibrio vulnificus or thebacteria Vibrio cholerae. The probes of the present invention areuniquely made of very short pieces of DNA, so small that they can easilyenter a Vibrio vulnificus or Vibrio cholerae cell, respectively.

The DNA probes of the instant invention are amazingly versatile in thatthey can be mixed with for example, water, food such as fish orshellfish, blood, feces or other type of samples to be tested. Toidentify the bacteria, a labeled moiety, such as a dye, a biotin or aradioisotope molecule, is attached to one end of each DNA probe. When afluorescent dye is selected and a light is shone on the sample (food,water, fish, shellfish, blood, feces, etc.), the fluorescent dye willglow only if the bacteria for which the DNA probe is specific arepresent. Quite amazingly, it is actually possible to see each tinybacterium glowing by using an epifluorescent microscope. And, becausethere is no need to culture or grow the bacteria beforehand or toamplify their genes, the tests of the present invention are simpler,much quicker and more reliable than the other kinds of test which havebeen available heretofore for detecting Vibrio vulnificus and Vibriocholerae.

The methods of the present invention involve fixation of whole bacterialcells from a mixed culture sample on, for example, membrane filters ormicroscope slides followed by hybridization with tagged species-specificprobe, and washing to remove excess or non-specifically bound probe. Thepresence or absence of Vibrio vulnificus or Vibrio cholerae cells isdirectly determined by, for example, epifluorescence microscopy when theDNA probes are labeled with a fluorescent dye.

Each probe of the instant invention is a single-strandedoligodeoxynucleotide sequence, approximately 15-20 bases in length,which specifically targets the ribosomal RNA (rRNA) component of theribosomes within Vibrio cholerae or Vibrio vulnificus cells.

While the DNA probes of the present invention typically include betweenabout 15 and about 20 nucleotides, it should nevertheless be understoodby those skilled in this art that the present invention contemplates DNAprobes of any size so long as the objectives of the present inventionare not defeated, i.e., the DNA probes are of a size sufficient so thatthey can enter the bacteria cells, and the DNA probes include aneffective number of nucleotides so that they can hybridize specificallywith the ribosomal RNA of either the bacteria Vibrio vulnificus or thebacteria Vibrio cholerae, respectively.

Accordingly, it can now be appreciated that the present invention isbelieved to provide a valuable and worldwide solution to the Vibriovulnificus and Vibrio cholerae art that has long sought effective andinexpensive means to quickly and reliably detect such pathogenicbacteria. As a result, the present invention can help to protect thepublic health, and perhaps prevent future epidemics, by its ability todetect infection in humans as well as in contaminated water and food atan early state.

The above features and advantages will be better understood withreference to the Detailed Description set out hereinbelow. It will alsobe understood that the biological materials of this invention areexemplary only and are not to be regarded as limitations of thisinvention.

DETAILED DESCRIPTION

By way of illustrating and providing a more complete appreciation of thepresent invention and many of the attendant advantages thereof, thefollowing detailed description is provided concerning the noveloligonucleotide probes, methods and kits.

The DNA probes of the present invention have been developed by PCRamplification and sequencing of the genes for the 16S and 23S rRNA ofVibrio vulnificus and Vibrio cholerae. The gene sequences were thenaligned with available sequences for other Vibrio and non-Vibrio rRNAgenes in order to locate regions of the genes which are unique to Vibriovulnificus or Vibrio cholerae. The DNA probes of the instant inventionwork by specifically binding to their complementary rRNA sequence on theribosome to form a DNA-RNA hybrid molecule.

The Vibrio vulnificus DNA probe sequences include:

SEQ ID NO:1

CGCTTCATTGAGCTA

SEQ ID NO:2

CGCTTCATTGAGCTAT

SEQ ID NO:3

CGCTTCATTGAGCTATG

SEQ ID NO:4

CGCTTCATTGAGCTATGT

SEQ ID NO:5

TGGCTTCATTGAGCTA

SEQ ID NO:6

TGGCTTCATTGAGCTAT

SEQ ID NO:7

TGGCTTCATTGAGCTATG

SEQ ID NO:8

TGGCTTCATTGAGCTATGT

SEQ ID NO:9

TTCGCTTCATTGAGCTAT

SEQ ID NO:10

TTCGCTTCATTGAGCTATG

SEQ ID NO:11

TTCGCTTCATTGAGCTATGT

The Vibrio cholerae DNA probe sequences include:

SEQ ID NO:12

GATTCCTAGGTTGAGCCCA

SEQ ID NO:13

GATTCCTAGGTTGAGCCCAG

The DNA probes of the present invention can be made in an automated DNAsynthesizer. An amino linker is attached to the 5′ end of the probe toallow covalent coupling of a fluorescent dye. Dye molecules that can beattached to the probes include the isothiocyanate or sulfonyl halideforms of fluorescein, a rhodamine and other fluorephores which arecommercially available. Fluorescently-tagged probes are purified on aSephadex G-25 size exclusion column followed by HPLC. At this point, theprobes are ready for use.

In addition to fluorescent dyes, the probes can be labeled with, forexample, a biotin, a radioisotope, or other tag molecules and thehybridization between the probe and its target rRNA molecule is detectedby means other than epifluorescence microscopy.

A method for detecting and identifying Vibrio vulnificus or Vibriocholerae using the fluorescently-tagged or labeled DNA probes involvesfixation of whole bacterial cells from a mixed culture sample (food,water, etc.) on a membrane filter or glass microscope slide followed byhybridization with the appropriate fluorescently-tagged,species-specific probe, and washing to remove excess or non-specificallybound probe. The presence or absence of Vibrio vulnificus or Vibriocholerae cells is directly determined by epifluorescence microscopy.

Samples of environmental or clinical origin (e.g. water, shellfish,blood) to be tested for the presence of these bacteria are adhered toeither glass microscope slides coated with gelatin or tonon-fluorescent, inorganic, 0.2 micron membrane filters and air dried.The sample is then fixed in a formaldehyde:phosphate buffered salinesolution (9:1), fixative solution A, for 20 minutes. This fixation stepis repeated with fresh fixative solution. (These fixation steps can beomitted if the original sample is fresh.) Next, the sample is fixed inmethanol:formaldehyde (9:1), fixative solution B, for 20 minutes, rinsedbriefly in distilled water and air dried.

A small volume of a hybridization solution or buffer containing theprobe is added to the fixed, dried sample. The hybridization solution orbuffer includes about 0.2% sodium dodecyl sulfate (SDS), sodiumchloride, sodium phosphate, EDTA (SSPE) (0.9 Molar sodium chloride, 0.06Molar sodium phosphate, 0.006 Molar EDTA), Denhardt's solution—1×, andabout 4 mcg/ml of double stranded heterologous DNA salmon sperm. Whenslides are selected, the hybridization solution or buffer should includeenough probe so that approximately 50 nanograms of probe available persmear per slide. When filters are selected, the hybridization solutionor buffer should include enough probe so that approximately 15 nanogramsof probe is available per filter. The slides/filters are incubated inthe dark at a specific temperature for about one to about two hours toallow hybridization between the probes and target rRNA. Thehybridization temperature for the Vibrio vulnificus probe is about 51°C. The hybridization temperature for the Vibrio cholerae probe is about57° C.

Washes to remove excess or non-specifically bound probes are performedat room temperature for a total of about 12 minutes followed by about a20 minute wash at the hybridization temperature with a solution of lowersalt content (wash solution B) than that of the first wash solution(wash solution A). Wash solution A includes 0.2% SDS and 6×SSPE (0.9Molar sodium chloride, 0.06 Molar sodium phosphate, and 0.006 MolarEDTA). Wash solution B includes 0.2% SDS and 1×SSPE (0.15 Molar sodiumchloride, 0.01 Molar sodium phosphate and 0.001 Molar EDTA).Slides/filters are rinsed briefly with distilled water, air dried in thedark, and viewed with an epifluorescence microscope.

To summarize a procedure of the present invention:

1. fix cells about (20-60 minutes);

2. hybridize with fluorescently-labeled probe solution (about 1-2hours);

3. Wash to remove non-specifically bound probe (about 35 minutes); and

4. view cells with epifluorescence microscope.

A typical kit of the present invention includes: fixative solutions A &B; fluorescently-labeled oligonucleotide DNA probe (5 nanograms/mlwater); hybridization buffer; wash solutions A & B; and non-fluorescentfilters or Teflon-coated microscope slides (with sample wells).

The DNA probes of the present invention have been laboratory tested andare believed to be species-specific. Fluorescently-taggedspecies-specific DNA probes of the present invention, which target rRNA,have been successfully used for both environmental research and clinicaldiagnostic purposes. The advantages of using such DNA probes fordetecting and identifying Vibrio vulnificus or Vibrio cholerae in eitherenvironments or clinical samples include the following: 1. rapid (oneday) diagnosis; 2. elimination of the need for prior culturing of thebacteria in test sample; 3. the probes are small enough to penetratewhole, intact, fixed bacterial cells (in situ hybridization); 4.elimination of the need to extract or amplify DNA; 5. detection oftarget organisms in a mixed bacterial sample; 6. fluorescent labelallows direct and immediate visualization of individual cells; 7.fluorescently-labeled probes are stable, sensitive and safe; and 8.methods utilize standard clinical and research laboratory equipment andrequires minimal technical expertise.

Applications of the probes contemplated by the present inventioninclude, for instance, clinical usage to rapidly diagnose humaninfections of Vibrio vulnificus or Vibrio cholerae, regulatory agencyuse to detect bacteria in water and food (especially important in oystermeat), and research usage for ecological studies of the seasonality anddistribution of these bacteria in the environment.

The present invention may, of course, be carried out in other specificways than those herein set forth without departing from the spirit andessential characteristics of the invention. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive and all changes coming within the meaning and equivalencyrange of the appended claims are intended to be embraced herein.

13 15 base pairs nucleic acid single linear DNA (genomic) not provided 1CGCTTCATTG AGCTA 15 16 base pairs nucleic acid single linear DNA(genomic) not provided 2 CGCTTCATTG AGCTAT 16 17 base pairs nucleic acidsingle linear DNA (genomic) not provided 3 CGCTTCATTG AGCTATG 17 18 basepairs nucleic acid single linear DNA (genomic) not provided 4 CGCTTCATTGAGCTATGT 18 16 base pairs nucleic acid single linear DNA (genomic) notprovided 5 TGGCTTCATT GAGCTA 16 17 base pairs nucleic acid single linearDNA (genomic) not provided 6 TGGCTTCATT GAGCTAT 17 18 base pairs nucleicacid single linear DNA (genomic) not provided 7 TGGCTTCATT GAGCTATG 1819 base pairs nucleic acid single linear DNA (genomic) not provided 8TGGCTTCATT GAGCTATGT 19 18 base pairs nucleic acid single linear DNA(genomic) not provided 9 TTCGCTTCAT TGAGCTAT 18 19 base pairs nucleicacid single linear DNA (genomic) not provided 10 TTCGCTTCAT TGAGCTATG 1920 base pairs nucleic acid single linear DNA (genomic) not provided 11TTCGCTTCAT TGAGCTATGT 20 19 base pairs nucleic acid single linear DNA(genomic) not provided 12 GATTCCTAGG TTGAGCCCA 19 20 base pairs nucleicacid single linear DNA (genomic) not provided 13 GATTCCTAGG TTGAGCCCAG20

Having described our invention, we claim:
 1. An oligonucleotide probespecific for Vibrio vulnificus, said oligonucleotide probe being capableof hybridizing specifically with a ribosomal RNA sequence selected fromthe group consisting of: CGCTTCATTGAGCTA (SEQ ID NO:1) CGCTTCATTGAGCTAT(SEQ ID NO:2) CGCTTCATTGAGCTATG (SEQ ID NO:3) CGCTTCATTGAGCTATGT (SEQ IDNO:4) TGGCTTCATTGAGCTA (SEQ ID NO:5) TGGCTTCATTGAGCTAT (SEQ ID NO:6)TGGCTTCATTGAGCTATG (SEQ ID NO:7) TGGCTTCATTGAGCTATGT (SEQ ID NO:8)TTCGCTTCATTGAGCTAT (SEQ ID NO:9) TTCGCTTCATTGAGCTATG (SEQ ID NO:10)TTCGCTTCATTGAGCTATGT (SEQ ID NO:11); and complementary sequencesthereto.
 2. An oligonucleotide probe as recited in claim 1, said probefurther including a label.
 3. An oligonucleotide probe as recited inclaim 2, said label being a fluorophore.
 4. An oligonucleotide probe asrecited in claim 3, said fluorophore being selected from the groupconsisting of a fluoroscein isothiocyanate, a fluoroscein sulfonylhalide and a rhodamine.
 5. An oligonucleotide probe as recited in claim2, said label being selected from the group consisting of biotin and aradioisotope.
 6. A method of identifying a Vibrio vulnificus, saidmethod comprising: hybridizing the labeled probe of claim 2 to ribosomalRNA of the Vibrio vulnificus; and detecting the labeled probe hybridizedto the ribosomal RNA to identify the Vibrio vulnificus.
 7. A method asrecited in claim 6, said method including the further step of: fixingthe Vibrio vulnificus prior to said hybridizing.
 8. A method as recitedin claim 7, wherein said detecting step comprises: detecting thehybridized labeled probe with a epifluorescence microscope.
 9. Anoligonucleotide probe specific for Vibrio cholerae, said oligonucleotideprobe having an oligonucleotide sequence for hybridizing specificallywith a ribosomal RNA segment, the ribosomal RNA segment comprising anucleotide sequence selected from the group consisting of:GATTCCTAGGTTGAGCCCA (SEQ ID NO: 12); GATTCCTAGGTTGAGCCCAG (SEQ ID NO:13); and complementary sequences thereto.
 10. An oligonucleotide probeas recited in claim 9, said probe further including a label.
 11. Anoligonucleotide probe as recited in claim 10, said label being afluorophore.
 12. An oligonucleotide probe as recited in claim 11, saidfluorophore being selected from the group consisting of a fluorosceinisothiocyanate, a fluoroscein sulphonyl halide and a rhodamine.
 13. Anoligonucleotide probe as recited in claim 10, said label being selectedfrom the group consisting of biotin and a radioisotope.
 14. A method ofidentifying a Vibrio cholerae, said method comprising: hybridizing thelabeled probe of claim 10 to ribosomal RNA of the Vibrio cholerae; anddetecting the labeled probe hybridized to the ribosomal RNA to identifythe Vibrio cholerae.
 15. A method as recited in claim 14, said methodincluding the further step of: fixing the Vibrio cholerae prior to saidhybridizing.
 16. A method as recited in claim 14, wherein said detectingstep comprises: detecting the hybridized labeled probe with anepifluorescence microscope.
 17. An oligonucleotide probe specific for aVibrio vulnificus bacterium, said oligonucleotide probe having asequence selected from the group consisting of: CGCTTCATTGAGCTA (SEQ IDNO:1) CGCTTCATTGAGCTAT (SEQ ID NO:2) CGCTTCATTGAGCTATG (SEQ ID NO:3)CGCTTCATTGAGCTATGT (SEQ ID NO:4) TGGCTTCATTGAGCTA (SEQ ID NO:5)TGGCTTCATTGAGCTAT (SEQ ID NO:6) TGGCTTCATTGAGCTATG (SEQ ID NO:7)TGGCTTCATTGAGCTATGT (SEQ ID NO:8) TTCGCTTCATTGAGCTAT (SEQ ID NO:9)TTCGCTTCATTGAGCTATG (SEQ ID NO:10) TTCGCTTCATTGAGCTATGT (SEQ ID NO:11);and complementary sequences thereto.
 18. An oligonucleotide probe asrecited in claim 17, said oligonucleotide probe further including alabel.
 19. An oligonucleotide probe as recited in claim 18, said labelbeing a fluorophore.
 20. An oligonucleotide probe as recited in claim19, said fluorophore being selected from the group consisting of afluoroscein isothiocyanate, a fluoroscein sulfonyl halide and arhodamine.
 21. An oligonucleotide probe as recited in claim 18, saidlabel being selected from the group consisting of biotin and aradioisotope.
 22. A method of identifying a Vibrio vulnificus bacterium,said method comprising: hybridizing the labeled probe of claim 18 to aregion of the 23S ribosomal RNA of the Vibrio vulnificus bacterium; anddetecting the labeled probe hybridized to the 23S ribosomal RNA toidentify the Vibrio vulnificus bacterium as a Vibrio vulniflcusbacterium.
 23. A method as recited in claim 22, said method includingthe further step of fixing a Vibrio vulnificus bacterium prior to saidhybridizing.
 24. A method as recited in claim 23, wherein said detectingstep comprises detecting the hybridized labeled probe with anepifluorescence microscope.
 25. A fixed Vibrio vulnificus bacteriumhaving hybridized thereto an oligonucleotide probe specific for a regionof the 23S ribosomal RNA of said Vibrio vulnificus bacterium, whereinthe oligonucleotide probe comprises a label, and wherein the region ofthe 23S ribosomal RNA comprises a sequence selected from the groupconsisting of: CGCTTCATTGAGCTA (SEQ ID NO:1) CGCTTCATTGAGCTAT (SEQ IDNO:2) CGCTTCATTGAGCTATG (SEQ ID NO:3) CGCTTCATTGAGCTATGT (SEQ ID NO:4)TGGCTTCATTGAGCTA (SEQ ID NO:5) TGGCTTCATTGAGCTAT (SEQ ID NO:6)TGGCTTCATTGAGCTATG (SEQ ID NO:7) TGGCTTCATTGAGCTATGT (SEQ ID NO:8)TTCGCTTCATTGAGCTAT (SEQ ID NO:9) TTCGCTTCATTGAGCTATG (SEQ ID NO:10)TTCGCTTCATTGAGCTATGT (SEQ ID NO:11); and complementary sequencesthereto.
 26. A fixed Vibrio vulnificus bacterium as recited in claim 25,said label being a fluorophore.
 27. A fixed Vibrio vulnificus bacteriumas recited in claim 26, said fluorophore being selected from the groupconsisting of a fluoroscein isothiocyanate, a fluoroscein sulfonylhalide and a rhodamine.
 28. A fixed Vibrio vulnificus bacterium asrecited in claim 25, said label being selected from the group consistingof biotin and a radioisotope.
 29. A fixed Vibrio vulnificus bacterium asrecited in claim 25, wherein said fixed Vibrio vulnificus bacterium isattached to a substrate selected from the group consisting of a slideand a membrane.
 30. A fixed Vibrio cholerae bacterium having hybridizedthereto a probe with a label, said labeled probe being specific for aregion of 23S ribosomal RNA of said Vibrio cholerae bacterium, theregion of 23S ribosomal RNA comprising a sequence selected from thegroup consisting of: GATTCCTAGGTTGAGCCCA (SEQ ID NO: 12);GATTCCTAGGTTGAGCCCAG (SEQ ID NO: 13); and complementary sequencesthereto.
 31. A fixed Vibrio cholerae bacterium as recited in claim 30,said label being a fluorophore.
 32. A fixed Vibrio cholerae bacterium asrecited in claim 31, said fluorophore being selected from the groupconsisting of a fluoroscein isothiocyanate, a fluoroscein sulphonylhalide and a rhodamine.
 33. A fixed Vibrio cholerae bacterium as recitedin claim 30, said label being selected from the group consisting ofbiotin and a radioisotope.
 34. A fixed Vibrio cholerae bacterium asrecited in claim 30, wherein said Vibrio cholerae bacterium is fixed toa substrate selected from the group consisting of a slide and amembrane.
 35. A kit for detecting a Vibrio vulnificus bacterium, saidkit comprising: an oligonucleotide probe as recited in claim 17, and atleast one component selected from the group consisting of a fixativesolution, hybridization buffer, wash solution, non-fluorescent filter,slide and membrane.
 36. A kit for detecting a Vibrio cholerae bacterium,said kit comprising: an oligonucleotide probe as recited in claim 10;and at least one component selected from the group consisting of afixative solution, hybridization buffer, wash solution, non-fluorescentfilter, slide and membrane.